Kinetic studies of HPr, HPr(H15D), HPr(H15E), and HPr(His approximately P) phosphorylation by the Streptococcus...

HPr is a central protein of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In streptococci, HPr can be phosphorylated at His(15) at the expense of PEP by enzyme I (EI) of the PTS, producing HPr(His approximately P). HPr can also be phosphorylated at Ser(46) by the ATP-dependent HPr(Ser) kinase/phosphorylase (HprK/P), producing HPr(Ser-P). Lastly, HPr can be phosphorylated on both residues, producing HPr(Ser-P)(His approximately P) (HPr-P2). We report here a study on the phosphorylation of Streptococcus salivarius HPr, HPr(H15D), HPr(H15E), and HPr(His approximately P) by HprK/P to assess the involvement of HprK/P in the synthesis of HPr-P2 in streptococcal cells. We first developed a spectrophotometric method for measuring HprK/P kinase activity. Using this assay, we found that the K(m) of HprK/P for HPr at pH 7.4 and 37 degrees C was approximately 110 muM, with a specificity constant (k(cat)/K(m)) of 1.7 x 10(4) M(-1) s(-1). The specificity constants for HPr(H15D) and HPr(H15E) were approximately 13 times lower. Kinetic studies conducted under conditions where HPr(His approximately P) was stable (i.e., pH 8.6 and 15 degrees C) showed that HPr(His approximately P) was a poorer substrate for HprK/P than HPr(H15D), the k(cat)/K(m) for HPr(H15D) and HPr(His approximately P) being approximately 9 and 26 times lower than that for HPr, respectively. Our results suggested that (i) the inefficiency of the phosphorylation of HPr(His approximately P) by HprK/P results from the presence of a negative charge at position 15 as well as from other structural elements and (ii) the contribution of streptococcal HprK/P to the synthesis of HPr-P2 in vivo is marginal.